u-0126 and Liver-Neoplasms

To investigate the role of the ROS/MAPK signaling axis in mediating the inhibitory effect of eriocitrin on proliferation and migration of hepatocellular carcinoma SMMC-7721 cells.. SMMC-7721 cells were treated with different concentrations of eriocitrin for 24 h, and the changes in cell viability were detected with CCK-8 assay. The migration and invasion abilities of the treated cells were evaluated using Transwell and scratch healing assays, the cell proliferation was assessed with colony-forming assay, and changes in nuclear morphology were observed with DAPI staining. Western blotting was performed to examine the changes in the expressions of E-cadherin, N-cadherin, MMP-2, MMP-9, PARP, Pro-caspase 3, pJNK, p-P38, and p-ERK. The effect of eriocitrin on PARP cleavage in SMMC-7721 cells pretreated with ERK, JNK and P38 inhibitors (U0126, SB203580 and SP600125, respectively) was detected using Western blotting. The effect of treatment with Nacetyl-cysteine (NAC, 30 μmol/L) and eriocitrin (100, 200, and 300 μg/mL), alone or in combination, on reactive oxygen species (ROS) levels in the cells was examined using a DCFH-DA fluorescent probe.. Eriocitrin can suppress the proliferation and migration and promote apoptosis of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPKs signaling pathway.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Poly(ADP-ribose) Polymerase Inhibitors; Reactive Oxygen Species

Hepatocellular carcinoma is one of the most common primary hepatic malignancy. Herein, a series of semisynthesized derivatives (2-30) of the natural product (+)-sclerotiorin (1) was prepared and evaluated the cytotoxic activities against six cancer cell lines. Among them, 3 and 5 were the most effective compounds against human hepatocellular carcinoma Bel-7402 cell line with IC

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzopyrans; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; Mice; Xenograft Model Antitumor Assays

It has been reported that miR-294 is highly expressed in hepatocellular carcinoma (HCC) tissues and cells. However, the potential role of miR-294 in the pathogenesis of HCC remains unclear. This study aimed to explore the role of miR-294 in HCC and the potential mechanism involved in this process.. Reverse transcription polymerase chain reaction was performed to determine the expression of miR-294 in HCC tissues and cell lines. Following the overexpression or knockdown of miR-294, the proliferation, migration, and invasion abilities of cells were determined using Cell Counting Kit-8 (CCK-8), wound healing and transwell assays, respectively. The phosphorylation of JNK and ERK was determined through western blotting. Furthermore, HCC cells were treated with JNK inhibitor SP600125 or ERK inhibitor U0126 and transfected with miR-294 mimics or negative control. Subsequently, the phosphorylation of JNK and ERK was evaluated and the proliferation, migration and invasion abilities of HCC cells were also determined.. The expression of miR-294 was significantly increased in HCC tissues and cell lines. Following the overexpression of miR-294, proliferation, migration, and invasion were promoted in the SSMC-7721 cell line, and the phosphorylation of JNK and ERK was increased, while silencing of miR-294 led to the opposite result. Use of the JNK or ERK inhibitor to treat SSMC-7721 cells transfected with miR-294 mimics decreased the phosphorylation of JNK and ERK and inhibited the proliferation, migration and invasion abilities of cells.. miR-294 is important for the development of HCC in terms of the biological activities of cells, and may be a novel therapeutic target for HCC.

Topics: Aged; Anthracenes; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; MAP Kinase Kinase 4; MAP Kinase Signaling System; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Nitriles; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Up-Regulation

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Chemotherapy is an alternative treatment for advanced HCCs, but chemo-resistance prevents cancer therapies from achieving stable and complete responses. Understanding the underlying mechanisms in chemo-resistance is critical to improve the efficacy of HCC.. The expression levels of Id-1 and CCN2 were detected in large cohorts of HCCs, and functional analyses of Id-1 and CCN2 were performed both in vitro and in vivo. cDNA microarrays were performed to evaluate the alterations of expression profiling of HCC cells with overexpression of CCN2. Finally, the role of downstream signaling of MAPK/Id-1 signaling pathway in oxaliplatin resistance were also explored.. The increased expression of Id-1 and CCN2 were closely related to oxaliplatin resistance in HCC. Upregulation of CCN2 and Id-1 was independently associated with shorter survival and increased recurrence in HCC patients, and significantly enhanced oxaliplatin resistance and promoted lung metastasis in vivo, whereas knock-down of their expression significantly reversed the chemo-resistance and inhibited HCC cell stemness. cDNA microarrays and PCR revealed that Id-1 and MAPK pathway were the downstream signaling of CCN2. CCN2 significantly enhanced oxaliplatin resistance by activating the MAPK/Id-1 signaling pathway, and Id-1 could upregulate CCN2 in a positive feedback manner.. CCN2/MAPK/Id-1 loop feedback amplification is involved in oxaliplatin resistance, and the combination of oxaliplatin with inhibitor of CCN2 or MAPK signaling could provide a promising approach to ameliorating oxaliplatin resistance in HCC.

Topics: Adult; Aged; Animals; Antineoplastic Agents; Biomarkers, Tumor; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Connective Tissue Growth Factor; Down-Regulation; Drug Resistance, Neoplasm; Feedback; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Liver Neoplasms; Male; MAP Kinase Signaling System; Mice, Nude; Middle Aged; Mitogen-Activated Protein Kinase 1; Neoplasm Recurrence, Local; Neoplasm Transplantation; Neoplastic Stem Cells; Nitriles; Oxaliplatin; Protein Kinase Inhibitors; RNA, Messenger; Sorafenib; Up-Regulation

It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing.. We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation.. Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

Topics: Animals; Benzimidazoles; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; CRISPR-Cas Systems; Cytoskeletal Proteins; DNA, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Neurofibromatosis 1; Genome-Wide Association Study; Hepatocytes; High-Throughput Nucleotide Sequencing; HMGA Proteins; HMGA2 Protein; Humans; Immunohistochemistry; Liver Neoplasms; Liver Neoplasms, Experimental; Membrane Glycoproteins; Mice; Mice, Knockout; Mice, Nude; Mitogen-Activated Protein Kinases; Nerve Tissue Proteins; Niacinamide; Nitriles; Phenylurea Compounds; Prognosis; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Pyridones; Pyrimidinones; ras Proteins; Real-Time Polymerase Chain Reaction; Receptors, Cell Surface; Sequence Analysis, DNA; Sorafenib; Survival Analysis; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The role of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) inhibiting migration in hepatocellular carcinoma (HCC) remains unknown. Expression of EFEMP1 in HCC cell lines were quantified by western blotting and real-time PCR. The role of EFEMP1 in HCC cell migration was explored in vitro via siRNA and adding purified EFEMP1 protein. The associated molecule expression was detected by western blotting after downregulation of EFEMP1 and also tested by immunohistochemistry. Eight pairs of HCC non-HCC liver samples and 215 HCC samples were subjected to immunohistochemistry. EFEMP1 was highly expressed in 7,721 and HepG2 HCC cell lines while HuH7 HCC cell line expressed the lowest level of EFEMP1 compared with the others. Downregulating EFEMP1 by siRNA markedly increased the migration ability of HCC cells while adding purified EFEMP1 protein inhibited HCC cell migration. Downregulation of EFEMP1 increased the expression of ERK1/2, MMP2 and MMP9. Furthermore, U0126 (a highly selective and potent inhibitor of pERK1/2) could abrogate the migration ability enhanced by siRNA. Accordingly, MMP2 and MMP9 were inversely expressed with EFEMP1 expression by immunohistochemistry. EFEMP1 downregulated in HCC tissues, and lower EFEMP1 expression was significantly associated with HCC patients with ascites (P=0.050), vascular invasion (P=0.044), poorer differentiation (P=0.002) and higher clinical stage (P=0.003).

Topics: Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Down-Regulation; Extracellular Matrix Proteins; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Immunohistochemistry; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Nitriles; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Signal Transduction

Hepatocellular carcinoma (HCC) is one of the most common malignant human tumors worldwide, but no effective therapeutic options are currently available. The cancer stem cell (CSC) has proven to play a central role in the development, metastasis, and recurrence of HCC. In this study, we report a dual functional mitogen-activated protein kinase inhibitor (U0126)-based therapy for treating both bulk HCC and HCC CSCs, using poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles as the drug carrier. It is demonstrated that nanoparticle encapsulation enhanced the cell uptake of U0126 in HCC CSCs and that enhanced endocytosis lead to augmented cytotoxicity of U0126 in HCC CSCs. Moreover, the nanoparticle encapsulation increased the inhibition of self-renewal capability, prolonged the circulation time, and increased the tumor accumulation of U0126 when compared with the use of the free inhibitor. The systemic delivery of U0126 remarkably enhanced the suppression of tumor development with decreased CSCs in the HepG2 xenograft simultaneously with reduced systemic toxicity.

Topics: Animals; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Carriers; Endocytosis; Enzyme Inhibitors; Female; Hep G2 Cells; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Transplantation; Nitriles; Polyethylene Glycols; Stem Cell Transplantation

Sorafenib is an inhibitor of a number of intracellular signaling kinases with antiproliferative, anti‑angiogenic and pro‑apoptotic effects in tumor cells. Sorafenib has been used in the therapy of advanced renal cell carcinoma. In the present study, using two human non‑small cell lung cancer (NSCLC)cell lines, A549 and NCI‑H1975, the effects of sorafenib on proliferation, apoptosis and intracellular signaling were systematically characterized. The results revealed that at a low concentration (5 µM) and early time point (6 h), sorafenib is capable of significantly stimulating proliferation of A549 cells, but not NCI‑H1975 cells. In addition, the comparison of the two cell lines revealed different cell cycle redistribution and apoptotic susceptibility to sorafenib at this concentration and time point. Western blot analysis revealed that sorafenib upregulated the expression of cyclin D1 and cyclin‑dependent kinase 2 and downregulated the expression of BAX at this specific point. Furthermore, sorafenib was confirmed to regulate the expression of cyclin D1 and apoptosis‑associated proteins through the regulation of extracellular signal‑regulated kinase 1/2 phosphorylation in A549 cells. These findings suggest that, although sorafenib has the potential for use in the treatment of renal cell carcinoma, this compound may also activate NSCLC cells at a specific time point.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; MAP Kinase Kinase Kinases; Niacinamide; Nitriles; Phenylurea Compounds; Phosphorylation; Signal Transduction; Sorafenib

Epidermal growth factor (EGF) and their receptor (EGFR) play an important role in the development of cancer proliferation, and metastasis, although the mechanism remains unclear. The present study aimed at investigating the role of EGF-EGFR signalling pathway in the development of human hepatocellular carcinoma (HCC) inflammatory environment. Gene profiles of inflammatory cytokines from HCC were measured. Cell bio-behaviours of HCC with low or high metastasis were detected by the live cell monitoring system. Cell proliferation was measured by CCK8. The protein level of CXCL5 and CXCL8 was measured by ELISA. The phosphorylation of PI3K, ERK, MAPK was measured by western blot. EGF significantly induced cell proliferation in HepG2 cells, but not in HCCLM3 cells. EGF prompted the cell movement in both HepG2 and HCCLM3 and regulated the production of CXCL5 and CXCL8 from HCC, which were inhibited by EGFR inhibitor, Erk inhibitor (U0126), or PI3K inhibitors (BEZ-235 and SHBM1009). HCC proliferation, metastasis and production of inflammatory cytokines were regulated via EGF-EGFR signal pathways. CXCL5 could interact with CXCL8, possibly by CXCR2 or the cross-talk between CXCR2 and EGFR. EGF-EGFR signaling pathway can be the potential target of therapies for HCC.

Topics: Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CXCL5; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Inflammation; Interleukin-8; Liver Neoplasms; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinolines; Receptor Cross-Talk; Receptors, Interleukin-8B; Signal Transduction; Tumor Microenvironment

Sirt6, a member of the mammalian sirtuin family, is a protein that is located in the nucleus and is an NAD+‑dependent deacetylase important in the control of metabolic activity and genome stability. Recently, several studies have demonstrated the potential role of Sirt6 in tumor biology; however, the role of Sirt6 in hepatocellular carcinoma (HCC) remains unclear. In the present study, Sirt6 protein expression was found to be downregulated in human HCC tissue compared with adjacent normal tissue. Knockdown of Sirt6 promoted growth of the HepG2 HCC cell line, whereas overexpression of Sirt6 inhibited the growth of HepG2 cells. Overexpression of Sirt6 induced apoptosis in HepG2 cells, which was demonstrated by a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay and cleaved caspase-3 immunoblotting. Furthermore, overexpression of Sirt6 decreased intracellular reactive oxygen species and superoxide anion levels. Finally, overexpression of Sirt6 inhibited phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), and blocking the ERK1/2 pathway with chemical-specific inhibitor U0126, attenuated the tumor suppressive effect of overexpression of Sirt6. Collectively, these data suggest that Sirt6 is a tumor suppressor in HCC cells and may be a promising therapeutic target in HCC.

Topics: Apoptosis; Butadienes; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; Down-Regulation; HEK293 Cells; Hep G2 Cells; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirtuins

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed and associated with the prognosis of HCC. However, the functions of Gal-3 in HCC cells are not fully understood. To address the function of Gal-3 in HCC cells, we used small interfering RNA (siRNA) to knock down Gal-3 expression in HepG2, an HCC cell line. We found that in vitro the silencing of Gal-3 decreased the proliferative activity, colony formation ability, migratory and invasive potential of HepG2 cells. The silencing of Gal-3 significantly decreased the mRNA and protein levels of urokinase-type plasminogen activator receptor (uPAR) as well as uPAR's downstream signaling transduction pathway, including phosphorylation of AKT. Furthermore, the downregulation of Gal-3 by siRNA resulted in significantly decreased activity of the MEK/ERK signaling pathway, and the treatment of HepG2 cells with MEK/ERK inhibitor U0126 significantly reduced the mRNA and protein levels of uPAR. Taken together, our results suggest that Gal-3 modulates uPAR expression via the MEK/ERK pathway, and that Gal-3 may be a potential therapeutic target for the treatment of HCC.

Topics: Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Galectin 3; Gene Knockdown Techniques; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Neoplasm Invasiveness; Nitriles; Receptors, Urokinase Plasminogen Activator; RNA, Small Interfering

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide and is highly resistant to chemotherapy. Yes-associated protein (YAP) is the downstream effector of the Hippo signaling pathway, which is frequently overexpressed in many types of cancers. Amplification of the YAP gene and overexpression of YAP in HCC have previously been reported to contribute to hepatocyte malignant transformation and tumor progression. In this study, we aimed to investigate the potential role of YAP in HCC chemoresistance. Overexpression of YAP resulted in resistance against doxorubicin-induced apoptosis in HCC cell lines, whereas suppression of the endogenous YAP expression by RNA interference demonstrated the reverse effect. Western blotting revealed that, following exposure to doxorubicin, YAP-overexpressing cells exhibited decreased cleaved PARP, increased phosphorylation of Akt and ERK1/2, and elevated Bcl-xL expression in comparison to the vector control. Inhibition of YAP expression sensitized HCC cells to doxorubicin, by exhibiting increased cleaved PARP, decreased levels of phosphorylated Akt, phosphorylated ERK1/2 and Bcl-xL expression. In addition, pretreatment with the MEK1/2 inhibitor U0126 but not the PI3-K inhibitor LY294002 significantly enhanced doxorubicin-induced apoptosis and decreased Bcl-xL expression in YAP-overexpressing HCC cells. Our data provide evidence that overexpression of YAP plays an important role in conferring doxorubicin resistance to HCC, which is at least partially mediated by YAP-induced activation of the MAP kinase pathway. Targeting YAP may be a promising adjunct for overcoming doxorubicin resistance in HCC.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Apoptosis; bcl-X Protein; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromones; Doxorubicin; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Nitriles; Phosphoproteins; Phosphorylation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Transcription Factors; YAP-Signaling Proteins

The rate of morbidity and mortality of hepatocellular carcinoma (HCC) in Taiwan has not lessened because of difficulty in treating tumor metastasis. Norcantharidin (NCTD) is currently used as an anticancer drug for hepatoma, breast cancer, and colorectal adenocarcinoma. NCTD possesses various biological anticancer activities, including apoptosis. However, detailed effects and molecular mechanisms of NCTD on metastasis are unclear. Thus, HCC cells were subjected to treatment with NCTD and then analyzed to determine the effects of NCTD on cell metastasis.. Modified Boyden chamber assays revealed that NCTD treatment inhibited cell migration and invasion capacities of HCC cells substantially. Results of zymography and western blotting showed that activities and protein levels of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (u-PA) were inhibited by NCTD. Western blot analysis showed that NCTD inhibits phosphorylation of ERK1/2. Testing of mRNA level, quantitative real-time PCR, and promoter assays evaluated the inhibitory effects of NCTD on MMP-9 and u-PA expression in HCC cells. The chromatin immunoprecipitation (ChIP) assay for analyzing the genomic DNA sequences bound to these proteins was reactive to the transcription protein nuclear factor (NF)-kappaB, which was inhibited by NCTD. The expression of NF-kappa B was measured by western blot analysis, which revealed decreased nuclear-factor DNA-binding activity after NCTD treatment.. NCTD inhibited MMP-9 and u-PA expression through the phosphorylation of ERK1/2 and NF-kappaB signaling pathway which serves as a powerful chemopreventive agent in HCC cell metastasis.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Survival; Hepatocytes; Humans; Liver Neoplasms; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Nitriles; Phosphatidylinositol 3-Kinases; Plasminogen Activator Inhibitor 1; Proto-Oncogene Proteins c-akt; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Transcription, Genetic; Urokinase-Type Plasminogen Activator; Wound Healing

To investigate the role of Hedgehog (Hh) signaling pathway in the invasion and metastasis of human hepatocellular carcinoma (HCC).. Eighty six HCC tissues samples and HCC cell line Bel-7402 were examined. The protein expression of sonic hedgehog (Shh), nuclear glioma-associated oncogene-1 (Gli1), MMP-9 and p-ERK1/2 in HCC was analyzed using immunohistochemistry and Western blot analysis. Boyden chamber assay and wound-healing assay were used to quantify the invasion and metastasis of Bel-7402 cells.. In 86 HCC tissue samples, the positive ratio of Shh and nucleus Gli1 was 67.44% (58/86) and 60.47% (52/86), respectively; the expression of nucleus Gli1 was correlated with the tumor pathological grade (P=0.034), and with the ability of the tumor to invade and metastasize (P=0.001); the expression of nucleus Gli1 was also correlated with p-ERK1/2 (P=0.031) and with MMP-9 (P=0.034). Neither Shh, nor nucleus Gli1 was observed in normal liver tissue. KAAD-cyclopamine (KAAD-cyc), a specific inhibitor of the Hh pathway, at the concentrations of 1 and 4 μmol/L inhibited the invasion and migration of Bel-7402 cells and decreased the expression of Gli1 in nucleus and MMP-9, p-ERK1/2 proteins in Bel-7402 cells. On the other hand, Shh, a ligand of the Hh pathway, at the concentration of 0.5 μg/mL produced opposite effects. The MAPK pathway inhibitors U0126 and PD98059 at the concentrations of 5 and 10 μmol/L inhibited invasion and metastasis of Bel-7402 cells induced by Shh, and decreased the expression of p-ERK1/2 and MMP-9. However, U0126 and PD98059 had no effect on the expression of Gli1.. Hh signaling pathway mediates invasion and metastasis of human HCC by up-regulating the protein expression of MMP-9 via ERK pathway.

Topics: Adult; Aged; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cinnamates; Dose-Response Relationship, Drug; Female; Flavonoids; Hedgehog Proteins; Humans; Immunohistochemistry; Liver Neoplasms; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Grading; Neoplasm Invasiveness; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1

Farnesoid X receptor (FXR), a pivotal factor maintaining bile acid homeostasis, has been recently shown to be a critical factor required for liver regeneration. The elucidation of the mechanism how FXR controls the proliferation of hepatocellular carcinoma cells is useful to establish the therapy for liver cancer. Here, we show that FXR plays a crucial role in the proliferation of human hepatocellular carcinoma cell line, HepG2, Huh7 and HLE. The treatment of HepG2 with FXR siRNA elevates the level of p16/INK4a expression resulting in the inhibition of cell proliferation. By contrast, FXR activation reduces p16/INK4a expression and stimulates the cell proliferation. The ectopic expression of the active form of Ras that causes strong activation of extracellular signal-regulated kinase (ERK) leads to the decrease in FXR expression, suggesting that FXR expression is negatively regulated via Ras/ERK pathway. The elevation of p16/INK4a expression and the inhibition of cell proliferation by FXR knockdown are also observed in Huh7 and HLE. In this study, we have suggested a novel mechanism by which hepatocellular carcinoma cell proliferation is regulated: FXR stimulates cell proliferation by suppressing the p16/INK4a expression, whereas Ras/ERK pathway down-regulates the FXR expression, leading to the suppressed cell proliferation in hepatocellular carcinoma cell lines.

Topics: Butadienes; Carcinoma, Hepatocellular; Cell Nucleus Shape; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p16; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hep G2 Cells; Hepatocyte Growth Factor; Hepatocyte Nuclear Factor 4; Humans; Liver Neoplasms; MAP Kinase Signaling System; Nitriles; Proto-Oncogene Proteins p21(ras); Receptors, Cytoplasmic and Nuclear; RNA, Small Interfering

Chronic ethanol intake is a significant risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). The effects of ethanol on extracellular signal-regulated kinase (ERK) activation, transforming growth factor alpha (TGF-alpha), and HCC growth were examined in this study.. HepG2, SKHep, Hep3B human HCC cells, or normal human hepatocytes were treated with ethanol (0-100 mM), exogenous TGF-alpha, TGF-alpha neutralization antibody or the MEK inhibitor U0126. TGF-alpha levels were quantified by ELISA. Growth was determined by trypan blue-excluded cell counts. Cell cycle phase distribution was determined by flow cytometry. Protein expression was determined by Western blot.. Ethanol treatment (10-40 mM) increased ERK activation in HepG2 and SKHep HCC cells but not in Hep3B or human hepatocyte cells. Growth increased in HepG2 (174 +/- 29%, P < 0.05) and SKHep (149 +/- 12%, P < 0.05) cells in response to ethanol treatment. Correspondingly, ethanol increased S phase distribution in these cells. U0126 suppressed ethanol-induced growth increases. Ethanol treatment for 24 h also raised TGF-alpha levels in HepG2 cells (118%-198%) and SKHep cells (112%-177%). Exogenous administration of recombinant TGF-alpha mimicked the ethanol-induced growth in HepG2 and SKHep cells; TGF-alpha neutralization antibody effectively abrogated this effect. The TGF-a neutralization antibody also prevented ERK activation by ethanol in HepG2 cells.. These data demonstrate that clinically relevant doses of ethanol stimulate ERK-dependent proliferation of HCC cells. Ethanol up-regulates TGF-alpha levels in HCC cells and enhances growth through cell cycles changes, which appear to be mediated through TGF-alpha-MEK-ERK signaling. Ethanol-MEK signaling in normal hepatocytes is absent, suggesting that ethanol promotion of HCC growth may in part depend upon the acquisition of cancer-specific signaling by hepatocytes.

Topics: Antibodies; Butadienes; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Central Nervous System Depressants; Enzyme Inhibitors; Ethanol; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; MAP Kinase Kinase 1; MAP Kinase Signaling System; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Transforming Growth Factor alpha

The MAPK MEK/ERK pathway is often upregulated in cancer cells and represents an attractive target for development of anticancer drugs. Only few data concerning the specific functions of ERK1 and 2 are reported in the literature. In this report, we investigated the specific role of ERK1 and 2 in liver tumor growth both in vitro and in vivo. DNA synthesis and cells in S phase analysed by flow cytometry, correlated with strong inhibition of Cdk1 and cyclin E levels, are strongly reduced after exposure to the MEK inhibitor, U0126. We obtained a significant reduction of colony formation in soft agar assays and a reduction in the size of tumor xenografts in nude mice treated with U0126. Then, we could specifically abolished ERK1 or 2 expression by small-interfering RNA (siRNA) and demonstrated that ERK2 knockdown but not ERK1 interferes with the process of replication. Moreover, we found that colony formation and tumor growth in vivo were significantly inhibited by targeting ERK2 using stable chemically modified siRNA. Taken together, our results emphasize the importance of the MEK/ERK pathway in liver cancer cell growth in vitro and in vivo and argue for a crucial role of ERK2 in this regulation.

Topics: Animals; Butadienes; Carcinoma, Hepatocellular; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin E; Enzyme Inhibitors; Female; Flow Cytometry; Humans; Immunoblotting; In Vitro Techniques; Liver Neoplasms; MAP Kinase Kinase Kinase 1; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Oncogene Proteins; Rats; RNA, Small Interfering; Transfection; Xenograft Model Antitumor Assays

Hepatocellular cancer (HCC) is a leading cause of cancer-related death worldwide. Historically, doxorubicin (DOX) has been widely used against unresectable HCC with variable response rates.. We hypothesized that DOX combined with mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (MEK-ERK) targeted therapy may provide enhanced anti-cancer effects. Human HCC cell lines (HepG2, Hep3B) were treated with DOX and MEK enzyme inhibitors, U0126 or PD184161, alone or in combination. Growth, apoptosis, and ERK expression/MEK activity were respectively determined by proliferation assay, DNA fragmentation enzyme-linked immunoassay or fluorochrome inhibitor of caspases, and Western blot.. DOX (0.01-1 microM) decreased cell proliferation in Hep3B cells (IC(50) approximately 0.12 microM) at 48 to 72 h; DOX was less effective in HepG2 cells (IC(50) approximately 0.25 microM). At early time points (30 min) after DOX treatment of Hep3B cells, MEK activity was unchanged at low doses and decreased at higher doses; after 24 h, phospho-ERK levels increased at higher doses. Contrarily, in HepG2 cells, DOX caused a sustained, dose-dependent increase in phospho-ERK levels at early and late time points. The MEK inhibitor U0126 decreased phospho-ERK in both HCC lines. In contrast to DOX, HepG2 cells were more sensitive than Hep3B cells to U0126. The combination of DOX with U0126 (or PD184161) resulted in greater inhibition of proliferation in HepG2 but not in Hep3B cells. This effect may be mediated in part by enhanced apoptosis.. The effect of DOX on early and late induction of MEK activity predicts its chemotherapeutic response in HCC. Furthermore, this effect may also determine the utility of MEK inhibitor combination treatment.

Topics: Aniline Compounds; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Enzyme Inhibitors; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation

Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway.. HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins.. Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells can correct the defect in VLDL assembly leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK-ERK cascade in VLDL assembly in the HepG2 model. Modulation of this pathway in primary hepatocytes also regulates apoB secretion and appears to alter the formation of VLDL-1 sized particles.

Topics: Animals; Apolipoprotein B-100; Butadienes; Cell Line, Tumor; Cricetinae; Diacylglycerol O-Acyltransferase; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Hepatoblastoma; Hepatocytes; Humans; Lipoproteins, VLDL; Liver Neoplasms; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mesocricetus; Nitriles; Oleic Acid; Particle Size; Rats; RNA, Messenger; Signal Transduction; Triglycerides

Sensitivity to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. However, it is unknown if TRAIL cytotoxic signaling engages the lysosomal pathway of cell death. Our aim, therefore, was to ascertain if TRAIL killing involves lysosomal permeabilization. TRAIL-induced apoptosis of hepatocellular carcinoma cells (HuH-7, Hep3B) was associated with lysosomal permeabilization, as demonstrated by redistribution of the lysosomal protease cathepsin B into the cytosol. Pharmacological and short hairpin RNA-targeted inhibition of cathepsin B reduced apoptosis. Because cellular FLICE-inhibitory protein (cFLIP) inhibits TRAIL-induced cell death and is frequently overexpressed by human cancers, the ability of cFLIP to prevent lysosomal permeabilization during TRAIL treatment was examined. Enforced long-form cFLIP (cFLIP(L)) expression reduced release of cathepsin B from lysosomes and attenuated apoptosis. cFLIP(L) overexpression was also associated with robust p42/44 MAPK activation following exposure to TRAIL. In contrast, cFLIP(L) overexpression attenuated p38 MAPK activation and had no significant effect on JNK and NF-kappaB activation. Inhibition of p42/44 MAPK by PD98059 restored TRAIL-mediated lysosomal permeabilization and apoptosis in cFLIP-overexpressing cells. In conclusion, these results demonstrate that lysosomal permeabilization contributes to TRAIL-induced apoptosis of hepatocellular carcinoma cells and suggest that cFLIP(L) cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown.

Topics: Apoptosis; Butadienes; Carcinoma, Hepatocellular; CASP8 and FADD-Like Apoptosis Regulating Protein; Cathepsins; Cell Line, Tumor; Flavonoids; Humans; Imidazoles; Liver Neoplasms; Lysosomes; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; p38 Mitogen-Activated Protein Kinases; Permeability; Protein Transport; Pyridines; TNF-Related Apoptosis-Inducing Ligand

Motility and invasiveness events require specific intracellular signaling cascade activations. In cancer liver cells, one of these mechanisms could involve the MAPK MEK/ERK cascade activation which has been shown over expressed and activated in hepatocellular carcinoma. To study whether the MEK/ERK cascade is involved in the motility of HCC, we examined the effect of MEK inhibitor and ERK2 silencing using monolayer wound-healing assays and fluoroblock invasion systems. Evidence was provided that the MAPK cascade is a key transduction pathway which controls HCC cells motility and invasiveness. We could disconnect proliferation to motility using mitomycin C and we established that RNAi-mediated inhibition of ERK2 led to strongly reduced cell motility. To improve our understanding, we analysed the regulation and the role of urokinase receptor (uPAR) in this process. We provided evidence that uPAR was under a MEK/ERK dependent mechanism and blocking uPAR activity using specific antagonist or inhibiting its expression by RNA interference which resulted in complete inhibition of motility. Moreover, we found in MAPK inhibited cultures and in uPAR silencing cells that p70S6K phosphorylation on residue Thr-389 was significantly reduced, whereas Ser-421/Thr-424 phosphorylation did not change. We highlighted that the FRAP/mTOR pathway did not affect motility and Thr-389 phosphorylation. Furthermore, we demonstrated that p70S6K inhibition by RNA interference completely inhibited hepatocarcinoma cell motility. Therefore, targeting uPAR and/or MEK/ERK/S6K by RNA interference could be a major therapeutic strategy for the future treatment of invasive hepatocarcinoma cells.

Topics: Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitomycin; Neoplasm Invasiveness; Nitriles; Peptides, Cyclic; Phosphorylation; Protein Kinase Inhibitors; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Time Factors; Wound Healing

The molecular mechanisms behind the anti-neoplastic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are not completely understood and cannot be explained by the inhibition of the cyclooxygenase (COX) enzymes COX-1 and COX-2 alone. We previously reported that both the selective COX-1 inhibitor SC-560 and the selective COX-2 inhibitor CAY10404 exhibit anti-tumor effects in human hepatoma cells. NSAID inhibitors have many COX-independent actions and, among others, the mitogen-activated protein kinase (MAPK) pathways are targets for NSAIDs. Here, we examined the role of MEK/ERK1/2 signaling in the anti-neoplastic effects of both selective COX-1 and COX-2 inhibitors in two human hepatoma cell lines. Treatment of hepatoma cells with the selective COX-1 inhibitor SC-560, as well as with the selective COX-2 inhibitor CAY10404, was associated with activation of ERK1/2 in a time- and dose-dependent manner. Treatment with COX-1 and COX-2 inhibitors in the presence of the selective MEK1/2 inhibitor U0126 effectively suppressed ERK1/2 activation and combinations of either SC-560 or CAY10404 with U0126 resulted in synergistic effects on cell growth inhibition and induction of apoptosis. In HuH-6 hepatoma cells the combination-induced apoptosis was associated with caspase-9 and -3 activation, PARP cleavage, release of cytochrome c from the mitochondria into the cytosol and down-regulation of survivin and beta-catenin levels. In conclusion, our study showed that growth inhibitory concentrations of selective COX-1 and COX-2 inhibitors increased ERK1/2 phosphorylation in hepatoma cells, and that inhibition of the MEK/ERK signaling pathway potentiates the antitumor activity of both types of inhibitors. Therefore, our results provide preclinical support for a combined chemotherapeutic approach with selective NSAIDs and MEK inhibitors for the treatment of hepatocellular carcinoma.

Topics: Apoptosis; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; Caspases; Cell Proliferation; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Enzyme Inhibitors; Flow Cytometry; Humans; Isoxazoles; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Pyrazoles; Sulfones; Time Factors; Tumor Cells, Cultured

The MEK-ERK growth signaling pathway is important in human hepatocellular carcinoma (HCC). To evaluate the targeting of this pathway in HCC, we characterized a novel, orally-active MEK inhibitor, PD184161, using human HCC cells (HepG2, Hep3B, PLC, and SKHep) and in vivo human tumor xenografts. PD184161 inhibited MEK activity (IC50 = 10-100 nM) in a time- and concentration-dependent manner more effectively than PD098059 or U0126. PD184161 inhibited cell proliferation and induced apoptosis at concentrations of > or = 1.0 microM in a time- and concentration-dependent manner. In vivo, tumor xenograft P-ERK levels were significantly reduced 3 to 12 hours after an oral dose of PD184161 (P < .05). Contrarily, tumor xenograft P-ERK levels following long-term (24 days) daily dosing of PD184161 were refractory to this signaling effect. PD184161 significantly suppressed tumor engraftment and initial growth (P < .0001); however, established tumors were not significantly affected. In conclusion, PD184161 has antitumor effects in HCC in vitro and in vivo that appear to correlate with suppression of MEK activity. These studies demonstrate that PD184161 is unable to suppress MEK activity in HCC xenografts in the long term. Thus, we speculate that the degree of success of MEK targeted treatment in HCC and other cancers may, in part, depend on the discovery of mechanisms governing MEK inhibitor signaling resistance.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Benzamides; Butadienes; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Humans; Liver Neoplasms; MAP Kinase Kinase Kinases; Mice; Mice, Nude; Neoplasm Transplantation; Nitriles

We previously showed that prolonged and strong ERK phosphorylation induced by Compound 5 (Cpd 5), a Cdc25A protein phosphatase inhibitor, was involved in its mechanism of cell growth inhibition. To study the relationship between ERK phosphorylation and cell growth inhibition, we used Cpd 5 as a tool to investigate ERK-regulated c-Myc expression in Hep3B hepatoma cells. We found that ERK phosphorylation caused by Cpd 5 induced c-Myc phosphorylation, but suppressed c-Myc expression at the mRNA and protein levels. Furthermore, Cpd 5 inhibited c-Myc transcriptional activity and DNA binding ability, and this inhibition was antagonized by ERK kinase (MEK) inhibitor U-0126, implying that the ERK pathway was involved in regulating c-Myc expression. Since the participation of c-Myc protein in transcription requires its dimerization with Max protein, we examined the Myc-Max association in Cpd 5-treated cells and found that Cpd 5 suppressed Myc-Max dimerization. Transfection of Hep3B cells with mutated ERK (T188A/Y190F), which has lost its dual-phosphorylation sites, attenuated the actions of Cpd 5 on Myc-Max association. To further demonstrate whether Myc phosphorylation by Cpd 5-induced ERK activation was able to directly regulate c-myc gene expression, a chromatin immunoprecipitation (ChIP) assay was used to examine the binding of phospho-Myc to the c-myc promoter region. We found that phospho-Myc induced by Cpd 5 had lost its ability to bind to the c-myc promoter, whereas MEK inhibitor U-0126 antagonized this inhibitory effect. These data suggest that an increase in c-Myc phosphorylation in response to prolonged ERK phosphorylation negatively auto-regulates c-Myc gene expression, leading to the suppression of its target gene expression and cell cycle block.

Topics: Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Liver Neoplasms; Nitriles; Oligonucleotide Array Sequence Analysis; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-myc; RNA, Messenger; Signal Transduction; Transcription, Genetic; Transfection; Vitamin K

Vitamin K2 (VK2) is an anti-proliferative agent toward a variety of cancer including hepatocellular carcinoma (HCC). Because the growth inhibitory effect of VK2 to HCC has not been established yet, we investigated it in HCC cells in vitro. VK2 inhibited growth of Hep3B, but not of HepG2, HLF, and Huh6. VK2 induced the cell cycle arrest at the G1 phase and involvement of apoptosis was suggested because the sub-G1 fraction appeared in flow cytometric analysis and nuclear condensation and fragmentation appeared after VK2 treatment. VK2 activated extracellular signal-regulated kinase (ERK)1/2 in a mitogen-activated ERK-regulating kinase (MEK)-dependent manner in Hep3B and Huh6, but not in HepG2 and HLF. When ERK1/2 was inhibited by U0126, apoptosis by VK2 in Hep3B, but not in Huh6, was significantly enhanced. However, Western blot analysis revealed that neither apoptosis induction by VK2 nor enhancement of apoptosis by U0126 was mediated by caspase activation. These data demonstrated that VK2 induced apoptosis and activated the MEK/ERK1/2 signaling pathway in a cell-type specific manner, and a MEK inhibitor could augment the cell death in these cells.

Topics: Apoptosis; Butadienes; Carcinoma, Hepatocellular; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Drug Synergism; Enzyme Activation; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Protein Kinase Inhibitors; Vitamin K 2

Human hepatocellular carcinoma (HCC) is associated with increased expression and activity of mitogen-activated protein kinase (MAPK) signaling intermediates (ie, MEK, ERK).. We determined the effects of MEK-ERK signaling on proliferation, cell cycle, apoptosis, and tumorigenicity of HCC in vitro. HCC cell lines were treated with MEK enzyme-specific inhibitors, PD098059 and U0126, and ERK1,2 oligonucleotide antisense.. In the HCC cells examined, MEK inhibitors blocked ERK1,2 phosphorylation without a change in total ERK expression. ERK1,2 oligonucleotide antisense inhibited ERK1,2 protein expression. PD098059, U0126, and ERK1,2 oligonucleotide antisense each inhibited HCC cellular proliferation in a concentration-dependent manner. Cell cycle, apoptosis, and tumorigenicity were examined in Hep3B and HepG2 cell lines. MEK enzyme inhibition resulted in anticancer effects through cell cycle arrest, increased apoptosis, and decreased tumorigenicity in these cell lines. U0126 exhibited more potent inhibition of ERK1,2 phosphorylation and had more pronounced anticancer effects in both cell lines. Correspondingly, HepG2 cells, the cell line more sensitive to ERK1,2 phosphorylation inhibition, sustained more pronounced anticancer effects with treatment. But Hep3B cells were more sensitive to ERK1,2 antisense-mediated decreases in ERK1,2 protein expression and correspondingly, their growth was inhibited to a greater degree than the HepG2 cells. MEK enzyme inhibition had downstream effects on the expression of the antiapoptotic protein survivin in both cell lines.. These data suggest that there are multiple anticancer effects of blocking MEK-ERK signaling, and that these depend on both the susceptibility of the cells and the ability of the treatment to effect a selective block of MEK-ERK signaling in HCC cells.

Topics: Apoptosis; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; Cell Count; Cell Division; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Humans; Liver Neoplasms; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Oligonucleotides, Antisense; Phosphorylation; Signal Transduction; Tumor Cells, Cultured; Tumor Stem Cell Assay

Primary cancer of the gallbladder is not unusual. Most cases of gallbladder cancer are found at an advanced stage, accompanied by the invasion to the liver, metastases to the lymph nodes and distant organs, and peritoneal dissemination. In this study, we first examined the effect of mitogen-activated protein kinase kinase (MEK) inhibitors on the production of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), and tissue inhibitors of metalloproteinases (TIMPs) in a human gallbladder cancer cell line, NOZ cells in vitro. MEK inhibitors (PD98059 and U0126) inhibited the production of MMP-2, MMP-9 and high MW uPA, and upregulated TIMPs (TIMP-1, TIMP-2 and TIMP-3). Subsequently, we examined the effect of U0126 on invasion and metastasis of orthotopically inoculated NOZ cells in nude mice. Direct liver invasion by cancer cells was detected in all of the mice in the control group, but in only one mouse in the U0126-treated group. Most of the primary tumors in the U0126-treated group expanded to the liver, but did not invade into the liver. Vessel invasion in the liver was evident in 4 out of 5 mice in the control group, but in only one mouse in the U0126-treated group. Lymph node metastases and peritoneal dissemination were recognized in all of the mice in both groups. All 5 mice in the U0126-treated group, and 4 out of 5 mice in the vehicle control group, had metastases in the lungs. The present results suggest that a MEK inhibitor, U0126, prolonged the survival of the mice with NOZ tumor by inhibiting direct liver invasion and vessel invasion of the cancer cells via down-regulation of the matrix degrading ability of the cancer cells.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Flavonoids; Gallbladder Neoplasms; Humans; Liver; Liver Neoplasms; Lymphatic Metastasis; MAP Kinase Kinase 1; Mice; Mice, Nude; Models, Anatomic; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Nitriles; Urokinase-Type Plasminogen Activator

Cyclooxygenase-2 (COX-2) and ERK-MAPK mitogenic signaling pathways are important in human hepatocellular carcinoma. We investigated the effect of COX-2 inhibition on ERK-MAPK signaling and the effect of combining MEK (MAPK kinase) and COX-2 inhibitors in human hepatocellular carcinoma in vitro. COX and ERK expression were determined by immunoblot in HepG2 and Hep3B cells. COX-2 and MEK activity were determined by prostaglandin E(2) assay and phosphospecific immunoblot, respectively. Cell growth was determined by cell proliferation and cell counts. Apoptosis was determined by DNA fragmentation enzyme-linked immunosorbent assay and flow cytometry. Cell cycle was determined by flow cytometry. HepG2 and Hep3B cells do not express COX-1 or COX-2. Correspondingly, basal and agonist (arachidonic acid, lipopolysaccharide)-stimulated COX-2 activity is undetectable. Treatment of HepG2 and Hep3B cells with NS398 resulted in an increase in ERK1/2 phosphorylation (MEK activity) in a concentration-dependent fashion (NS398, 1 to 100 micromol/L). Treatment with the COX-2 inhibitor NS398 in the presence of U0126 (MEK inhibitor) effectively suppressed ERK1/2 phosphorylation as determined by phosphospecific ERK1/2 immunoblot. Total ERK1/2 and COX-2 were unchanged with NS398 and U0126 treatments. In HepG2 cells, NS398 (1 to 100 micromol/L) decreased apoptosis as determined by DNA fragmentation enzyme-linked immunosorbent assay. Relative apoptosis was increased with U0126 alone or in combination with NS398 (9 to 10 times the control value), eliminating the anti-apoptotic effect of NS398. In Hep3B cells, apoptosis was unchanged with NS398 (1 to 50 micromol/L) or U0126 (1 to 10 micromol/L) alone. The combination of NS398 and U0126 in Hep3B cells resulted in a synergistic increase in apoptosis (10 times the control value). Relative apoptosis in both cell lines strongly correlated with changes in the expression of the antiapoptotic protein Bcl-xL. Cellular growth was assessed by colorimetric proliferation assay and cell counts. HepG2 and Hep3B cells had concentration-dependent inhibition of cell growth with NS398 or U0126 treatment alone. The combination of NS398 and U0126 resulted in complementary inhibitory effects on growth. Growth inhibitory effects in HepG2 and Hep3B cells with combination treatment appear to be, in part, secondary to the induction of G(0)/G(1) and G(2)/M cell cycle arrest, respectively, as determined by flow cytometry. Despite differential signaling in HepG

Topics: Adenocarcinoma; Apoptosis; Butadienes; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Drug Synergism; Enzyme Inhibitors; Humans; Isoenzymes; Liver Neoplasms; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Nitrobenzenes; Pancreatic Neoplasms; Prostaglandin-Endoperoxide Synthases; Signal Transduction; Sulfonamides